A long non-coding RNA lnc-PMIF and its small interfering RNA and application

A long-chain non-coding, small interference technology, applied to long-chain non-coding RNAlnc-PMIF and its small interfering RNA and its application fields

Active Publication Date: 2021-06-29
NORTHWESTERN POLYTECHNICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this study was limited to iPSC-derived osteoblasts from patients with Li-Fraumeni syndrome, and did not study osteoblasts under normal non-pathological phenomena (such as aging, menopause, long-term bed rest, etc.) (Lee DF, et al. Modeling familial cancer with induced pluripotent stem cells, Cell. 2015 Apr 9; 161(2):240-254)

Method used

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  • A long non-coding RNA lnc-PMIF and its small interfering RNA and application
  • A long non-coding RNA lnc-PMIF and its small interfering RNA and application
  • A long non-coding RNA lnc-PMIF and its small interfering RNA and application

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Experimental program
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Embodiment 1

[0034] Illumina sequencing technology platform to detect the expression of lncRNA in the bone-forming cells of the aged osteoporosis mouse model and the ovariectomized osteoporosis mouse model; it shows the difference in the expression of lncRNA in the bone-forming cells during the aging process .

[0035] (1) To establish a mouse model of aging osteoporosis:

[0036] C57BL / 6 experimental mice were selected and raised to the age of 6 months (control group) and 18 months (old group).

[0037] (2) Execute the mice, collect the femur and tibia, separate the bone marrow, place the bone marrow in osteoblast conditioned medium, and store in 5% CO 2 Incubate at 37°C under conditions.

[0038] (3) Total RNA was extracted from the cells by the Trizol method, and the sample purity (A260 / A280) was detected by a spectrophotometer;

[0039] (4) Agilent 2100RNA Nano 6000Assay Kit (Agilent Technologies, CA, USA) detects the integrity and concentration of RNA samples;

[0040] (5) Take 3 ...

Embodiment 2

[0044] Acquisition of lnc-PMIF full-length cDNA sequence information:

[0045] The present embodiment provides the method for obtaining the full-length cDNA sequence of long-chain non-coding RNA lnc-PMIF, which specifically includes the following steps:

[0046] Step 1. Total RNA was extracted from the mouse-derived MC3T3-E1 cell line using the Trizol method, and ribosomal RNA (rRNA) in the sample was removed. Based on the Illumina sequencing technology platform, the paired-end sequencing method was used to construct a strand-specific library for sequencing, and obtained lnc RNA-seq data;

[0047] Step 2. According to the lncRNA sequence information obtained by sequencing, design 3'RACE and 3'RACE primers as follows;

[0048] Table 1 RACE primers

[0049]

[0050] Step 3. Use the RACE kit 3'-Full RACE Core Set with PrimeScript TM RTase (Takara) and 5'-Full RACE Kit with TAP (Takara) were used for 3'RACE and 5'RACE respectively to obtain RACE products;

[0051] Step 4, ...

Embodiment 3

[0054] Using qPCR technology, it was found that lnc-PMIF was highly expressed in the bone-forming cell samples of the aged osteoporosis mouse model and the ovariectomized (OVX) osteoporosis mouse model.

[0055] (1) Design and prepare specific primers for lnc-PMIF, the sequences are shown in Table 2;

[0056] (2) Establish an aging mouse model of osteoporosis:

[0057] C57BL / 6 experimental mice were selected and raised to the age of 6 months (control group) and 18 months (old group). The mice were sacrificed, and femur and bone marrow cell samples were collected.

[0058] (3) Establish OVX osteoporosis mouse model:

[0059] C57BL / 6 experimental female mice were selected, and the waist was shaved after anesthesia as the operation area. The ovaries and uterine horns were found on both sides of the dorsal midline by 0.5 cm respectively, and the ovaries and uterine horns were found with tweezers. A surrounding mass of fatty tissue about the size of an ovary. During the experime...

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Abstract

The invention discloses a long-chain non-coding RNA lnc-PMIF and a small interfering RNA of lnc-PMIF. In addition, the present invention also discloses the application of the long-chain non-coding RNA lnc-PMIF in the preparation or screening of products for the diagnosis of human skeletal system diseases, and the application of small interfering RNA in the preparation of drugs or pharmaceutical compositions for the treatment of human skeletal system diseases . The present invention verifies the function of lnc-PMIF by designing a small interfering RNA sequence, and proves that lnc-PMIF can inhibit osteoblast migration through HuR, and the lnc-PMIF sequence can be used for the diagnosis of skeletal system diseases or as a therapeutic target; the designed small interference The RNA sequence can be used to prepare medicines and pharmaceutical compositions for treating skeletal system diseases, including human osteoporosis, femoral head necrosis, joint degeneration, scoliosis and other skeletal system diseases.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and medicine, and in particular relates to a long-chain non-coding RNA lnc-PMIF, a small interfering RNA and applications thereof. Background technique [0002] Non-coding RNA (Non-coding RNA) refers to RNA that does not code for protein. These include microRNA (miRNA) and long non-coding RNA (long non-coding RNA, lncRNA for short). The common feature of these RNAs is that they can be transcribed from the genome, but do not encode proteins, and perform their respective biological functions at the RNA level. Among them, lncRNA is a kind of eukaryotic endogenous RNA longer than 200nt, widely distributed in eukaryotic organisms, and plays an important regulatory role in various physiological and pathological processes such as cell differentiation, organ development and disease occurrence. Osteoblasts are the main functional cells for bone formation. As people age, bone development also ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/6883A61K31/713A61P19/10A61P19/02A61P19/08
CPCA61K31/713A61P19/02A61P19/08A61P19/10C12N15/113C12N2310/141C12N2320/30C12Q1/6883C12Q2600/158C12Q2600/178
Inventor 骞爱荣李迪杰杨超飞田野印崇陈志浩赵帆张茹赵欣罗晓庆
Owner NORTHWESTERN POLYTECHNICAL UNIV
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